Fornesic Medicine

The techniques of medical science applied to assist in the resolution of crimes, legal disputes etc. constitutes forensic medicine. Establishing the identity of victim (e.g of murder, accidents etc) is critical to solving the problems of crimes cases. DNA fingerprinting or DNA profiling, is a highly sensitive and extremely versatile approach to this problem.
Preparation of DNA sample: Common sources of DNA include blood, sermen solid tissures blood stains, semen stains, hair roots etc. The specimens should be stored under clean, dry, cool condition to minimize contamination by microorganisms and DNA degradation due to DNase .
The DNA is prepared as follows.
Cells lysed with the enzyme proteinase K which also digests protein residual protein may be separated using the detergent sodium dodecyl sulphate RNA can be eliminated by phenol pure high molecules weight DNA may be prepared using alternative protocol.
In case of mixed specimens, suitable separation procedures should be used in separate the constituent cells. For example viginal swabs from rape types can be extracted separately following specific procedures.
The degree of DNA degradation is assessed as follows soutnern blottin is combined with species specific probes to establish DNA identity of human origin or otherwise DNA degradation is estimated by electrophoresis in agarose gel staining with ethidium bromide and observation under UV light high molecular weight DNA appears as single large band whereas degraded DNA forms a large smear.
DNA quantity in the sample is determined by absorbance 260 nm agarose gel electrophoresis with DNA preparations of known quantity and staining with ethidium bromide to assess the intensity of stain taken and fixing the DNA on membrane filter and hybridizing with a labelled highly repetitive primate specific locus.
Approach of DNA analysis: Many polymorphic loci composed of tandem repeats of 2-80 base pair the number of repeats varying from 1-100 VNTR and polymorphic for the number of tandem repeats while minivariant repeats show polymorphism for base pair composition at specific sites within each repeat. This variation at one or more loci may be analyzed by RFLP or PCR can also be used to analyze polymorphic site that may or may not be located in tandem repeat units. The DNA sample or the PCR products may be analyzed by the following approaches.
VNTR analysis: The sample DNA is used for RFLP analysis this requires high molecular weight DNA PCR products are used to develop RAPD pattern this can use much smaller even somewhat degraded DNA preparations.
Dot blot Analysis: The DNA samples is subjected to PCR to amplify the loci polymorphism at which is be determined. The PCR products are fixed to membrane and hybridized with allele specific probes.
Base sequence Analysis: The base sequence of specific loci in the region of DNA can be determined variable region by DNA sequencing and used for preparing DNA profiles.

Gene Therapy

Human beings from more than 5000 different diseases caused by single gene mutations. e.g cystic fibrosis acatalasia Huntingtons chorea. Tay sach's disease Lesch-Nyhan syndrome sickle cell anemia, mitral stenosis, Hunter's syndrome haemophilia several forms of muscular dystrophy etc. In addition many common disorder like cancer, hypertension, atherosclerosis and mental illness seem to have genetic components.
Gene therapy may be defined in broad general terms as follows. Introduction of a normal functional gene into cells which contains the defective allele of concerned gene with the objective of correcting a genetic disorder or an acquired disorder. Application of gene therapy involves the following basic development in genetics molecular biology and biotechnology (1) determination of the role its products in health and disease (2) identification of the gene that play the key role in the development of genetic disorder (3) development of an approach for gene therapy.
The candidate disorder for gene therapy are selected on the basis of this following criteria (1) the disease should be life- threatening (2) gene responsible for the disease has been cloned (3) a precise regulation of the gene should not be required and (4) a suitable delivery system should be available.

Types of Gene Therapy

Gene therapy may be classified into the two types (1) germ line gene therapy and (2) somatic cell lgene therapy. In case of Germ line gene therapy germ cells sperms or eggs integrated into their genomes. Therefore, the change dur to which are ordinarily integrated into their genomes. Therefore, the change due to therapy is heritable and passed onto later generations. This approach theoretically is highly effective in counteracting the genetic disorders. However, this option is at least for the present for application in human beings for a variety of technical and ethical reasons.
In somatic cell gene therapy the gene is introduced only in somatic cells, especially of those tissues in which expression of the concerned gene is critical for health. Expression of the introduced gene relives symptoms of the disorder but this effect is not heritable as it does not involve the germline. Somatic cell therapy is the only feasible option, and clinical triasl have already started most for the treatment of cancer and blood disorders. This approach is divided into two categories.
(1) Augmentation therapy
(2) targetted gene transfer.

Augmentation Therapy

In this type of somatic cel gene therapy the functional gene introduced in addition so the defective gene endogenous to the cells the modified cell contain both the defective well as the normal copies of the gene. There are two general approaches to augmentation therapy. The first approach was used in the two general approaches to augmentation therapy. The first approach was used into the first two patients on whom gene therapy was attempted to correct the genetic disorder called Servere Combined Immune Deficiency syndrom produced by adenosine deaminase deficiency (1) normal gene copies was produced by cloning and the (2) packing into a defective retrovirus most of the viral genes were replaced by ADA gene(3) Lymphocytes were isolated from the patients and (4) the infected cells expressing the ADA gene were injected back the patients. The normal ADA gene was expressed the patients and ADA deficiency was portially corrected this resulted in an improvement in the patients immune system.
The variety of viral vectors have been used to deliver genes into the stem cells bone marrow cells cultured in vitro.
The second approach is the direct injection of DNA into the tissues either protein complexes into order to bring about receptor mediated transfer of DNA into specific tissue or even flaked DNA into muscle skin. Exciting result have been obtained with experimental hypercholesrolemia where IDI receptor levels have been augmented by injection of the gene as s sialoglycoprotein complex. THe problems in this approach as well related to the frequency of cells taking up and expressing the gene and more particularly the duration of expression. The gene delivery methods used for gene therapy can also be used for treatment of cancer. In case of cancer a toxin encoding gene can be delivered into the cancer cells. Similarly, appropriate interleukin genes can be delivered to boos the body defence mechanism.

Targetted Gene Transfer

Targetted gene transfer or gene targetting uses homologous recombination to repalce the endogenous gene with the functional introduced gene. The The first case of such gene transfer was used to disrupt the human B globin gene in cultured cells over through this approach more than 100 mammalian found through this sequence the site for recombination and is different from the gene to be introduced. Hence the sequence to be introduced is located in the inner region of the vector and is flanked by the sequences involved in recombination. A recombination of such a vector with its homologous cellular sequences produces duplication of the targetted sequences this is called insertional recombination. A strategy has been devised to modify only small sequence of the largest gene without the attendant gene duplication produced by insertional recombination. This approach, called in out method of gene targetting consists of the following two steps.
The first step called "in" steps, is targetted gene transfer using insertion vector the appropriately targetted cell will have gene duplication.
Second step termed as "out" step, depend on either intrachromosomal recombination or unequal sister chromatid exchange between homologous chromosome the recombination product of interest is chromosomes which has only a single and functional copy of the introduced gene.
The in-out strategy has been tested using the HGPRT gene. The gene was targetted into a mouse embryonic stem cell line subsequently it has been succsfully used with some other gene. This procedure is ideal for gene therapy.
Gene targettin is the strategy of choice for gene therapy for the following reasons.
(1) The targetted gene is changed in precise and specific manner.
(2) The introduced functional gene is placed in the same context it is flanked by the same DNA sequences as the replaced endogenous gene.
(3) No other gene of the genome is affected. The major limitation of the approach is the low frequency of homologous recombination this problem however is being removed by refinments of the teqchique. The feasibility of gene targetting has been demonstrated in number of different cell tyupes for several different genes. It is expected that targetted therapy would become a feasibility for many genes disease in the near future.

Drug Designing

This approach aims at designing drugs which specifically and selectively fit into the critical sites of the target molecules thereby inactivating the latter. The target molecule may be an enzyme concerned with either metabolism of DNA replication or hormone receptor or some other important molecule involved in a disease. A successful example of drug designing is provided by the drug proprandolol used for the treatment of heart attacks and hypertension was awarded J. Black the Nobel prize for physiology and Medicine in 1988 for this work Heart ailment are mainly due to an excess of the hormones norepineophrine and epinephrine, which act through two receptors called B propanolol blocks the B receptor thereby interfering with the action of these hormones.
Another drug called eimetidine blocks the H2 receptor in stomach lining of histamine histamine induces the release of HCl is stomach leading to the development of stomach ulcers. Cimetidine therefore, spacifically cures peptic ulcers by blocking the concerned receptor G Elison and G. Hitchings have used this approach to the useful drugs for the treatment of cancer gout, malaria and viral infections like herpes. Recently drug called azidohymidine has been developed for the treatment of AIDS this drug selectively inhibits the reverse transcriptase of HIV.
Another drug Ro-31-8959 designed to inhibit the HIV protease in stage 3 clinical trials using oral delivery inhibition of protease results in the production of immature non infections viral particles.

Disease Treatement

Treatment of diseases utilize a wide variety of preparations of both biological and abiological origins. The preparations of biological origin may either be crude ayuvedic medicines some allopathic drugs or purified to various degrees. Many of such compounds are obtained from plants but large number of them originate from micro organism cultured cells and recombinant organisms.
Micro-organism: A large number or pharmaceuticals originate from microorganisms they range from whole micro organism through biomass used as food supplements to a variety for highly valuable compounds like antibiotics, vitamins, enzymes, organic acids etc.
Plant Cell cultures: Some biochemicals of pharmaceutical value are produced by cultured plant cells e.g shikonin, berberine, ginseng biomass and taxol.
Animal cell culture: Cultured animal cells are the source of several compounds used in treatment of diseases e.g angiogenic factor, interlukin interferon.
Products from Recombinant Organisms: The product is obtained from nonrecombinant organisms are limited to their natureal capabilities. Genetic engineering has however, removed this limitation and genes from any organism can be transferred andexpressed into any other organisms.
Genetically Engineered Micro-organism: A large number of human genes encoding pharmaceutically valuable proteins have been cloned and expressed in micro-organisms. Initially E.coli was used as the host for obvious reasons of ease in cloning. But yeast is fast becoming the host of choice for production of recombinant proteins. Several of the recombinant proteins, used for treatment of diabetes dwarfism cancer interferons, interleukins granulocyte macrophage colony stimulating factors thromosis and AIDS.
Animal Cell Culture: More recently, cultured animal cells have been preferred for the expression of human genes encoding pharmaceutically valuable proteins. Some of these proteins have already been approved for therapeutic use tissue plasminogen activator erythroprotein and blood clotting factors the last two of the proteins have been approved for marketing in India.
Transgenic Plants: Plants are highly desirable in many of ways of commercial production of proteins of value. A large number of transgens encoding pharmaceutically valuable proteins have been expressed in plants. There is at least one example of commercial production of a recombinant proteins, hirudin, encoded by synthetic gene expressed in Brassica napus.
Advantage: Production or recombinant proteins of pharmaceutical value in microorganisms cultured animal cells or plants offers several important advantages over their conventional routes of productions.

Detection of Genetic Disease

In human there are hundred genetic which are mostly produced through recessive mutation. There is no cure to any of them except for the fast emerging option of gene therapy. Their incidence can be minimized an early detection of the afeicted which are then aborted. Therefore, when a woman gets pregnant the probability of her having child suffering from genetic disorder is estimated based on the histones of her and her husbands families further investigation is carried out for a clear cuts and specific diagnosis.
Obtaining Foetal Cells. Earlier foetal cells were obtained by amniocentesis withdrawal ofamionitic fluid with the help of hypodermic syringe. But amniocentesis is applicable only 18 weeks later after the pregnancy which is rather later of abortion. Therefore, foetal cells are now obtained from biopsies of trophoblastic villi which are an external part of human embryo and later form a part of the placenta. The biopsy is performed during 6 or 8 week of pregnancy an endoscope passed through the cervix of uterus usually provide 100 mg of pure foetal DNA.
Disease Detection.
The foetal cells are used for detection of the genetic disorders in one of the following ways.
(1) Determination of karyotype of cells provides information on various syndromes produced by gross chromosomal aberrations.
(2) Most of the genetic diseases produce defective protein or no enzymes many of these proteins have been identified and some of these can be assayed. The foetal cells are used to assay the concerned enzyme activities to detect such genetic disease. Atleast 35 genetic disease can be detected by assaying activities of specific enzymes. In case of some genetic diseases, the concerned gene mutation may alter the recognition site for restriction enzyme. The RFLP produced can be detected by Southern hybridization a sequence of the concerned gene is used as probe.
(3) A more general approach utilizes oligonucleotide probes representing the sequence altered by the gene mutation causing the genetic disease. Typically set of two separate probes are used for disease one probe is complementary to the normal sequence, while the other is complementary to the mutant sequence. The probes are radio labelled and used to probe Southern blost, under appropriate condition the probes can distinguish the normal and mutant DNA samples.

Autoantibodies

Autoantibodies are those antibodies that are specific to those antigens which are normally tolerated by the immmune syste and are typically constituents of cells and tissues of the animal in question such antigens are called autoantigens. Autoantibodies are produced in conditions of autoimmunity, which may be simply described an attack by the immune system on the host itself the antibodies are either cell specific or non organ specific they recognise variety of cellular and subcellular components including the components of replicon transcription RNA processing RNA translation and protein processing. The antigenic specific of antibodies can be useful in clinical diagnosis of the associated autoimmune disease.

Immuno- PCR

This techniques used PCR amplification of a marker DNA segment attached to an antibody for detection oaf the antigen for which this antibody is specific. The protocol of immuno-PCR may be stated in simple terms as follows.
(1) Test samples suspected of containing the antigen are added to microtitre plate wells and the antigen is immobilized on the surface of wells.
(2) The free binding sites on the microtire well surfaces are suitably blocked.
(3) The antibody specific to the antigen to be detected is added to the wells it forms antigen antibody complex, this occurs only in those wells where the antigen is present.
(4) The molecular linker add and bind the free region of the antibody A chimera is the most versatile molecular liker this chimaeric linker binds to the Fe domain of antibodies due to the protein A sequences. IT also binds to biotinylated DNA molecules due to the streptavidin mioety. The linker molecular is already complexed with the marker DNA molecule when it is added to the microtitre wells.
(5) A segment of the marker DNA is amplified by PCR.
The PCR products are analyzed by gel electrophoresis for a large scale application the PCR products can be labelled using flourochromes and happiens which permit their rapid even automated detection in the case of LCR.
The antigen antibody complex will be formed only in those microtitre wells which contain the target antigen. Therefore, PCR amplification will occur only in such wells in other products will contain the target antigen.
Immuno-PCR is highly sensitive it is several orders of magnitude more sensitive than ELISA. It can detect rare antigens and diagnose pathological condition much earlier it is extremely versatile that it can be applied to even single cells, can yield quantities estimates of the antigen and is amenable in addition is is relatively simple.

Ligase Chain Reaction

In ligase chain reaction theclinical sample is prepaed according to protocol to which liberates the DNA preseint in the sample the prepared clinical sample is added to reaction mixture containing thermostable DNA ligase a vast excess of two double strand oigonucleotide probes specific to the pathogen to be detected and NAD. The two probes are blunt ended represent contiguous segments of the pathogen genome and each of them long 15 to 30 pair long therefore, the target sequence long the target sequence must be specific to the pathogen to be detected therefore the reaction mixture is beatede in thermocycler to ensure strand separation of both the target DNA and the probes the temperature is then lowereed to 55 temperature to allow the probes to pair with the target DNA now joins the adjacent of one strand of probe of the complementary strands of the two probes are also similarly joined the product of ligation of the two probes is called amplicon.
THE second cycle of LCR is initiated through heating the reaction mixture 94 temperature. In this and subsequent cycles of LCR both the target DNA and the amplicon serve as targets for probes and a result for aomplification this leads to an exponential amplification of the amplication. The amplicons are detected by gel electrophoresis of the reaction mixture ethidium bromide staining and viewing under UV light.
LCR is highly efficient it deetects target molecules in sample having 200-300 targets. It is highly specific and rarely produces false positive signals which is in contrast to PCR. The LCR producere allows automated detection by employing flouresence or hapen labelled probes LCR has been used to detect wide variety infectious agents such as chlamydia trachomatis mycobacterium tuberculosis herpes simplex virus hepatitis B virus Hepatitis C virus etc.

Disease Diagnosis

It is very difficult of the disease iand its causal organism is to effective management and cure conventionally disease diagnosis is based on the following.
(1) Microscopy. The specimen are subjected to microscopic examination for detection of the causal organism for example stool examination for ova and cyst.
(2) Culture of the specimen on specific and selective media to allow specific pathogens to grow which are then tested for their susceptibility to various therapeutic agents for example antibodies.
(3) Immunological assay for specific antigens present on the surface of pathogens.
(4) Detection and measurement of the pathogen specific antibodies produced by the patient in response to the invasion by pathogen in case of viral infections.

These tests are tedious take long time culture methods may yield result immunological assays since they are based on polyclonal and some of them can not be applied in certain case for example antibody titre in case of latent viral infections. Novel diagnostic approaches have been by biotechnology which are precise and very rapid (1) probes and (2) monoclonal antibodies.

DNA Vaccines

Vaccines based on DNA are being developed and the results obatined influenza virus. The strategy of DNA vaccines is as follows the gene encoding the relevant immunogenic protein is isolated cloned and integrated into a suitable expression vector. This is as introduced into the individual to the immunised. THE gene is ultimately expressed in vaccinated individual and the immunogenic protein is expressed in sufficient invoke both humoral and cell mediated immunities. It may be pointed the various approaches for DNA vaccines are as follows.
(1) injection of pure DNA (2) use of vector adenovirus retrovirused E.coli salmonella typhimurium herpesviruseds for delivery of the gene (3) reimplanation of autologous cells into which the gene has been transferred and gun delivery of plasmid DNA which contains the gene in an expression casette.
In skeletal muscle ladthe injection of pure DNA or RNA leads to the uptake and expression of DNA in the muscle cells when gene encoding an immunogenic protein is so introduced its expression also results in immunizaion of the individuals. This approach has potential for delivery of DNA vaccines. The DNA most likely enters the skeletal muscle cells through transient discontinuties in their plasma lemma produced by stretching of the muscle cells during exercise.
To remove cells from the body of an individual into which the concerned immunogen encoding gene is introduced and expressed. These cells are then ireintroduced into the body of the individual in variety of ways. This approach although more cumbersome has the advantage of enabling control of the modified cells within contaniments.

A large number of gene encoding antigenic proteins have been integrated to the genome which was then used for vaccination. The antigen included viral protein like rabies virus glycoprotein herpes simplex virus B surface antigen vesicular stomatitis glycoprotein etc. These vaccince contained both hormonal and cellular immunities. THe recombinant V V vaccine against rabies is also highly and highly effective. Some antigen of HIV has been produced by recombinant V V into experimental animals and human volunteers these antigens induced detectable cellular immunity to the expressed HIV protein. There are several antigens encoding gens each from different pathogen may be incorporated into single V V genome. Such a recombinant V V vaccine will produce immunity to several diseases from a single inoculation such vaccines are called polyvalent vaccines. These is little data on the efficiency of such vaccines the use of DNA for immunization is often called genetic immunization.

Biotechnology and Health Care

In biotechnology originated for their potential applicatons in health care of both human and animal but primarily the former. It is in this sector that the contribution of biotechnology are most frequent more notable and that whole gamut of contributions in text of limited space these could be grouped under the following broad heads (1) disease prevention (2) therapeutic agents (3) fertility control of these categories bu citing appropriate examples. (4) forensic medicine is is aimed to highlight the major developments under each of these categories by citing appropriate examples.
Disease prevention vaccines. It is the most desirable most convenient and highly effective approach to health this achieved by vaccination or immunization using biological preparations called vaccines. Vaccines represent invaluable contribution biotechnology they provide protection against even such for which effective cures are not yet available.
An ideal vaccine should have the following features.
(1) It should not be tumerogenic or toxic or pathogen.
(2) It should have very low levels of side effects.
(3) It should not spread either within the vaccinated individual or to other individuals.
(4) It should be effective in producing long lasting hormonal and cellular immunities.
Conventional vaccines. Conventional vaccines consist of whole pathogenic organisms which may either by killed or live the virulence of pathogens is greatly reduced most viral vaccines conventional vaccine is highly effective having the following features
(1) In many case live vaccines have to be used since killed pathogen vaccines are ineffective.
(2) Live vaccines are generally based on cultured animal cells, hence expensive tissue culture set up is essential.
(3) Live vaccines are heat labile due to the pathogen inactivation by heat.
(4) Conventional vaccines carry a variable risk of disease development due to the occasional presence of active virus particles or reversion to virulence after replication in the vaccinated individual.
Purified antigen vaccines. These vaccine are based on purified antigens isolated from the concerned pathogen they donot contain the organism the risk of pathogenicity is avoided their cost is higher to the steps involved in purification and vaccince preparation and many of the isolated antigens are poorly immunogenic.
Many bacteria produce exotoxins which are highly immunogenic but these toxins produce toxic effects the intensity of which decreases with storage and this decline is accelerated by heat formaldehyde and other chemicals. Some toxic are ery good adjuvants increase the immunogenicity of other antigens for example diptheria toxoid for example the B polysaccharide of Haemophilus influenzae ispoorly immuogenic. But when the B polysaccharide is combined with the diptheria toxoid its immunogenicity in greatly increased in many cases such adjuvant activities can be used to great advantage since most of the isolated antigens from pathogens are poorly immunogenic.

ENZYME LINKED IMMUNO SORBENT ASSAY(ELISA)

An antibody (Ab) reacts with the concerned antigen (Ag) in a highly specific manner reacts only with that determinant or region of an antigen for which it is to produce an complex. When soluble protein react with the Ab Ag complex aggulatinates. In either case either the amount Ag of Ab complex formed or the rate of its formation is used to determine either quality of the antigen or the antibody involved in the interaction. The various assay used for the purpose are follows.
(1) preciptin reaction (2) the luchterlony electrophoresis are reaction in gels (3) aggulation reaction and (5) labelled antibody reactions.

Elisa Procedure: A generalized procedure and the basis principle of ELISA as follows. The antigen of interest in immobilized on the surface of the test tube petriplate or microtier well for convenient handling of large numbers of test sample these plate have allowed to react with the absorbed antigen unreacted molecules of the antibody are washed away leaving only the Ag-Ab complex. In secondary reaction antiglobulin react with the all the antibody is added into the vessel and allowed to react with the Ag-Ab complex. In an other approach unlabelled specific to antigen is immobilized in a micro titer well.

Probes

These are small nucleotide sequences useto to detect the presence of complementary sequences in nucelic acid samples. These are 15-30 base long this is achieved by permitting the probes to base pair with the sample nucleic acid and then identifying the samples that show base pairing with the probes. The detection of hybridzation is highly precise and extremely sensitive provided the probes are suitably labelled for an easy detection. The DNA and RNA both are used as a probes DNA probes are more convenient and preferable but double stranded DNA molecules can also be used RNA probes are ordinarily single stranded.
Preparation of probes Probes can be obtained by several ways some of the important ones are briefly described below.
(1) Highly purified mRNA can be used as a probe mRNA are naturally single stranded.
(2) Single strand RNA probes can be readily prepared by cloning the DNA seuence inserted into a special vector this vector has different and specific prokaryotic promoter on the two ends of the DNA insert.
(3) DNA segment isolated from the genome of an organism or cDNA molecules prepared by using mRNA can be cloned in E.coli different recombinant clones of this vector will yield probes complementary to one or the other strand of the cloned DNA segment.
(5) single strand cDNA probes can be prepared by limiting the copying of mRNA by reverse transcriptase to only one strand.

(6) PCR can be used to generate single stranded copies of DNA seuences this is called asymmetric PCR.

Western blotting

In western blotting technique the protein are transferred in nitrocellulose or nylon membrane The various steps of this techniques are described below.
(1) Proteins band separated to polyacrylamide gel electrophoresis.
(2) The protein bands are transferred onto nitrocelluolose or nylonmembrane initially this was achieved a capillary movement of buffer similar to southern blotting but now a days it is usually done by electrophoresis. Electrophoresis technique has been used in southern and nothern hybridization as well as buffer of low ionic strength and nylon membrane (since nucleic acid bind to nitrocellulose membrane only under condition of high ionic strength are used). The electrophoretic blotting both of proteins and nucleic acids is much faster and more efficient than capillary blotting.
(3) The specific protein are identified in two ways.
(1) for the specific antibodies used as a probes for detection of a specific anitgen.
(2) For the identification of glycoprotein lectin are used as a probes.

These probes may themselves be radioactives or radioactive molecule may be tagged to them. Often the idenitificaiton process is based on a sandwich reaction.

DNA sequencing

The determination of nucleotide or base sequence of DNA molecule is known as DNA sequencing. At present DNA sequencing is possible for only one to long DNA fragments. DNA sequencing has become feasible as a result following important developments (1) the availability of restriction endouncleases development of highly sensitive gel electrophoretic techniques which can separate fragments different by one nucleotide only (2) the gene cloning and PCR techniques large quantities of individual DNA fragments and (4) the development relatively easy and rapid DNA sequencing called Maxam and Gilbert procedure and (2) enzymatic procedure